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Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from ice crystallization and the freezing-induced denaturation of myofibrillar proteins, the latter of which has, however, not received much research focus. This review discusses the relationship between myofibrillar protein denaturation and water-holding capacity of meat in freezing-thawing with the aim to improve the understanding the relative importance of protein denaturation in the formation of thaw loss. The contribution of decreased pH and high ionic strength in the unfrozen water in freezing is emphasized and we hypothesize that these two factors are causing protein denaturation and conformational changes within muscle fibers, and consequently loss of water-holding capacity. Slow freezing produces more thaw loss than fast freezing, and this is discussed here in relation to the impacts on myofibrillar protein denaturation induced by the freezing rate.

This study aimed to investigate the effect of storage at -3 ?C on myofibrillar protein in fast or slow frozen pork. Five pork loins at 48 h post-mortem were subjected to either fast (cold metal plate/-80 ?C) or slow freezing (still air/-20 ?C) followed by storage at -3 ?C for 0, 1, 3, and 7 days before thawing. Freezing rate significantly influenced myofibrillar proteins within 3 days at -3 ?C, evidenced by higher thaw loss, higher surface hydrophobicity and reduced water-holding of myofibrils, and accelerated appearance of a myosin-4 fragment (160 kDa) in slow freezing. However, these observed differences disappeared after 7 days of storage at -3 ?C. The meat pH after thawing did not differ between fast and slow freezing rate. However, the pH values after thawing in both groups decreased with extended storage at -3 ?C. Our results suggest that the beneficial effects of fast freezing are gradually lost by holding at -3 ?C due to more extensive protein denaturation.

The aim of this study was to investigate the role of Ca

This study aimed to determine the effect of frozen-then-chilled storage on free Ca2+, proteolytic enzyme activity of calpains and the proteasome, water-holding capacity and shear force of porcine longissimus thoracis et lumborum muscle. Pork loins were subjected to either chilled storage at 2 +/- 1 degrees C for 1, 2, 4, 6 and 9 days, or frozen then chilled storage (20 +/- 1 degrees C for 1 week followed by thawing overnight). Free Ca2+ increased with chilled storage in the non-frozen group. Frozen-then-chilled storage increased free Ca2+ concentration, followed by a faster decrease of calpain-1 activity and activation of around 50% of calpain-2. Proteasome activity was reduced by around 40% following freezing-thawing. Purge loss increased and water-holding capacity of myofibrils decreased in the frozen-thawed group, suggesting considerable denaturation of myofibrillar proteins. Shear force was not affected by freezing-thawing, and we speculate that the tenderizing effect of calpain activation was counteracted by loss of proteasome activity and substantial exudate loss.

The aim of this study was to investigate the role of Ca2+ in the process of calpain-2 becoming associated to myofibrils and the potential of myofibril-bound calpain to degrade myofibrillar proteins. Different Ca2+ concentrations were applied to myofibrils mixed with partially purified calpain-2. Ca2+ induced binding of calpain to myofibrils in a concentration-dependent manner. The half-maximal Ca2+ requirements for binding of calpain-2 to myofibrils and for calpain-2 proteolysis of myofibrils were 0.60 mM and 0.29 mM, respectively. To investigate the proteolytic activity of myofibril-bound calpain, a mixture of myofibrils and calpain-2 was briefly incubated with Ca2+. Unbound calpain was removed by washing with a Ca2+-free buffer. The myofibril-bound calpain maintained proteolytic activity and degraded desmin when re-activated with Ca2+. In conclusion, the results suggest that an increase in Ca2+ will activate and induce binding of calpain to myofibrils. Subsequently, calpain is relatively tightly bound and proteolytically active.

The role of heat-denatured sarcoplasmic proteins in water-holding is not well understood. Here we propose a new hypothesis that in PSE-like conditions denatured sarcoplasmic proteins aggregate within and outside myofilaments, improving the water-holding of denatured myofibrils. The process is compartmentalized: 1) within the filaments the denatured sarcoplasmic proteins shrink the lattice space and water is expelled; and 2) between the myofibrils and in the extracellular space, the coagulated sarcoplasmic proteins trap the expelled water from interfilamental space. The effect of sarcoplasmic proteins on the water-holding of myofibrils following incubation for 1h at 21 to 44°C was investigated. Our results were consistent with the new hypothesis. Myofibrils without sarcoplasm had the poorest water-holding. With increasing amount of denatured sarcoplasmic proteins, the water-holding of heat-denatured myofibrils improved proportionally. X-ray diffraction was used to measure the lattice space between the filaments. Precipitated sarcoplasmic proteins shrank (P

Pork loins were stored at 5 °C for 14 days to investigate the effect of oxygen concentration in modified atmosphere packaging (MAP) on shear force and oxidation of lipids and proteins. The modified atmosphere contained 0 to 80% O2, 20% CO2, and balanced with N2. The results showed that shear force and thiobarbituric acid-reactive substances (TBARS) values increased with increasing oxygen concentration. Protein oxidation when measured as loss of free thiol groups, was greater in meat packaged under oxygen (20–80%). Myosin heavy chain (MHC) cross-linking, another marker of protein oxidation, was greater in MAP with 80% oxygen than 0% and 20% oxygen. Desmin degradation was not affected by the presence of oxygen, suggesting that the mechanism of oxygen-induced toughening of meat is through protein oxidation leading to cross-linking of structural proteins rather than through inactivation of proteolytic enzymes leading to reduced proteolysis.

Heat treatment of meat at temperatures between 50 and 65 °C, for extended periods of time, is known as low-temperature long-time (LTLT) cooking. This cooking method produces meat that has increased tenderness and better appearance than when cooked at higher temperatures. Public concerns regarding this method have focused on the ability to design heat treatments that can reach microbiological safety. The heat treatment induces modification of the meat structure and its constituents, which can explain the desirable eating quality traits obtained. Denaturation, aggregation, and degradation of myofibrillar, sarcoplasmic and connective tissue proteins occur depending on the combination of time and temperature during the heat treatment. The protein changes, especially in relation to collagen denaturation, along with proteolytic activity, have often been regarded to be the main contributors to the increased meat tenderness. The mechanisms involved and the possible contribution of other factors are reviewed and discussed.

The purpose of this study was to increase the knowledge on the relationship between proteolysis of myofibrillar proteins and the water-holding of meat. Myofibrils isolated from porcine longissimus thoracic et lumborum muscle were used as a model system. Myofibrils were incubated with either calpain-2, the proteasome or a lysosomal extract at 25 degrees C for 2 h. All three proteolytic systems improved the relative water-holding and generally there was a larger effect with increasing amount of enzymes in the incubation. The improved water-holding occurred in parallel to degradation of myofibrillar proteins. Desmin was degraded by calpain-2 as well as by lysosomal enzymes and a-actinin was released by the proteasome. We here propose a model in which degradation of proteins in and around the Z-disk allows overall swelling of the filament lattice and more specifically in the I band area. In conclusion, proteolytic degradation of myofibrillar proteins by calpain-2, the proteasome or lysosomal enzymes improves the water-holding of myofibrils.

The basic contractile unit of muscle, the sarcomere, will contract as the muscle goes into rigor post-mortem. Depending on the conditions, such as the rate of pH decline, the cooling rate and the mechanical restraints on the muscles, this longitudinal shortening will result in various post-mortem sarcomere lengths as well as lateral differences in the distances between the myosin and actin filaments. This shortening is underlying the phenomena described as rigor contraction, thaw rigor, cold shortening and heat shortening. The shortening in combination with the molecular architecture of the sarcomere as defined by the myosin filaments and their S-1 and S-2 units, the interaction with the actin filaments, and the boundaries formed by the 2-disks will subsequently influence basic meat quality traits including tenderness and water-holding capacity. Biochemical reactions from proteolysis and glycogen metabolism interrelate with the sarcomere length in a complex manner. The sarcomere length is also influencing the eating quality of cooked meat and the water-holding in meat products.

Patties were made from raw minced beef after storage for 6days in modified atmosphere (0, 20, 40, 60, and 80% oxygen) to study the combined effect of oxygen concentration and cooking temperature on hardness and color. Increased oxygen concentrations generally led to larger (P

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.

The effect of low temperature long time (LTLT) heat treatment at 48 °C, 53 °C, 58 °C, and 63 °C for T c (time to reach a core temperature equal to the water bath), T c + 5 h holding time, and T c + 17 h holding time was studied in Longissimus dorsi and Semitendinosus muscles from slaughter pigs and sows. Meat toughness (Warner–Bratzler Shear Force), cooking loss and color (Minolta L*a*b*-values) was measured and in the cooking loss the amount of heat-soluble collagen and activity of cathepsin B + L was determined. Decreasing shear force and increasing cooking loss during LTLT treatment was observed between 53 °C and 58 °C. Furthermore, increasing temperature from 53 °C to 58 °C and increasing time from T c to T c + 17 h increased the solubility of collagen. Residual activity of cathepsin B + L in LTLT treated pork was mainly affected by temperature, showing the highest activity at 58 °C and 63 °C.

A novel approach was applied in this study to directly evaluate the effect of muscle fiber type on postmortem protein degradation. Porcine muscle fibers were isolated from longissimus muscle at day 1, 3, and 6 postmortem. Fibers were sorted by immunochemical myosin heavy chain isoform typing. Western blot analysis of fibers pooled separately into type I or IIb showed that the relative amounts of 39- and 50-kDa desmin degradation fragments at day 6, and 28- to 31-kDa fragments of troponin T fast type isoform (fTnT) at day 1 and 6 postmortem were higher in type IIb than in type I fibers. At day 6 troponin T slow type isoform (sTnT) was less degraded than fTnT in type I fibers. These results indicated greater rate and extent of proteolysis in type IIb than in type I fibers and higher susceptibility of fTnT to proteolysis than that of sTnT isoform.

The objective of the present work was to characterize changes in calpain activity in pork post-mortem. Samples from pig M. longissimus dorsi and M. semimembranosus were collected three days post-mortem from 75 animals and analyzed with casein zymography. The results indicated post-mortem autolysis of m-calpain as two m-calpain bands were observed on the zymogram gel. Use of M. longissimus dorsi from three pigs collected at different times during storage further confirmed post-mortem autolysis of m-calpain. The activity of the autolyzed form of m-calpain was detectable at day 3 and further increased at day 6. The results also showed a decrease in the non-autolyzed m-calpain activity during post-mortem storage. Collectively, these results suggest that m-calpain is active post-mortem in porcine muscles.

The effect of proteolysis and heating on the mechanical properties of single muscle fibres isolated from bovine M. semitendinosus was investigated. Short incubations with μ-calpain (pH 7.5) and cathepsin B (pH 5.6) at room temperature reduced (P

This study investigated the influence of post-mortem pH decline on calpain activity and myofibrillar degradation. From 80 pigs, 30 Longissimus dorsi (LD) muscles were selected on the basis of pH values at 3 h post-mortem and classified into groups of 10 as fast, intermediate and slow pH decline. The rate of pH decline early post-mortem differed between the three groups, but the ultimate pH values were similar at 24 h. Calpain activity and autolysis from 1 to 72 h post-mortem were determined using casein zymography and studied in relation to myofibrillar fragmentation. Colour and drip loss were measured. A faster decrease in pH resulted in reduced level of μ-calpain activity and increased autolysis of the enzyme, and hence an earlier loss of activity due to activation of μ-calpain in muscles with a fast pH decline. Paralleling the μ-calpain activation in muscles with a fast pH decline a higher myofibril fragmentation at 24 h post-mortem was observed, which was no longer evident in the later phase of the tenderization process. In conclusion, the rate of early pH decline influenced μ-calpain activity and the rate but not the extent of myofibrillar degradation, suggesting an early effect of proteolysis on myofibril fragmentation that is reduced during ageing due to an earlier exhaustion of μ-calpain activity.

The aim was to investigate the effect of ageing large beef cuts directly in high oxygen modified atmosphere (MA) and how ageing time in vacuum influence meat quality when followed by retail packaging in high oxygen MA. Large cuts (10 cm long) of beef loin (LD) were aged for up to 25 days postmortem in different ageing systems at 4 °C. Ageing solely in high oxygen modified atmosphere (MA, 80% O2 + 20% CO2) for 5 or 10 days and ageing in vacuum for 5 or 15 days followed by high oxygen MA for 5 or 10 days were compared with ageing in vacuum for 5, 15 and 25 days. Warner–Bratzler shear force, purge and cooking losses, calpain activity and desmin and carbonyl contents were measured. Shear force decreased to the same level when ageing this large beef cut solely in high oxygen MA for 5 or 10 days as when ageing in vacuum. The activity of μ-calpain disappeared, the activity of m-calpain decreased and purge loss increased between 5 and 10 days, but cooking loss and the contents of desmin and carbonyls were unaffected. The ageing time in vacuum before packaging of this large beef cut in high oxygen MA did not affect the ultimate shear force. The m-calpain activity decreased and the content of carbonyls increased compared with solely in vacuum after 15 days of total ageing, but there was no difference in the content of desmin or cooking loss between these ageing systems at the same ageing time.

The degradation of bovine muscle proteins by proteasome and ubiquitous calpains was explored via 2D gel proteome analysis by inhibition of the physiological level of the proteases by specific inhibitors. The inhibition of the proteasome chymotrypsin- and trypsin-like activity results in the lack of degradation of several fragments of structural proteins such as actin, troponin T, myosin light chain and nebulin. In addition the degradation of several sarcoplasmatic proteins was eliminated when proteasome was inhibited. The inhibition of the ubiquitous calpain only resulted in minor changes in the degradation pattern, which might indicate that p94, which is not inhibited by calpastatin, is involved in the degradation post-mortem. The results of the present study indicate a sequential degradation of the structural proteins post-mortem, where calpain initiates the disruption and destabilisation of the myofibrillar structure, and thereby allows the proteasome to act.

The stability of μ-calpain, m-calpain and calpastatin activity during frozen storage of pork was studied in two experiments. In experiment 1, pork longissimus muscle was stored at either −20 or −80 °C, and the samples were assayed at 2–3 weeks interval for calpain activity and calpastatin activity using a m-calpain stock solution stored at 4 °C. No effects on calpain activity at either temperature were observed for up to 123 days of storage. Calpastatin activity was stable the first few weeks of storage, where after it decreased up to 143 days of storage independently of meat storage temperature. At day 143, calpastatin activity was also assayed using a newly purified stock solution of m-calpain giving a calpastatin activity equal to the activity measured day 0 using the original m-calpain stock solution. The m-calpain stock solution was unstable during storage at 4 °C and the activity decreased in a linear manner and was highly related to the observed decrease in calpastatin activity during storage. In experiment 2, meat was stored as in experiment 1 and was assayed at 2–3 week intervals for calpastatin activity using a m-calpain stock solution stored at either 4 or −80 °C. As in experiment 1, the measured activity of calpastatin decreased during storage using m-calpain stock solution stored at 4 °C and this decrease was highly correlated to the decrease in the activity of the m-calpain stock solution. The activity of the m-calpain stock solution stored at −80 °C was constant during storage period of 153 days and likewise was the calpastatin activity measured using this stock solution. The relation between measured calpastatin activity and storage time of m-calpain stock solution was tested by adding, to a calpastatin assay, up to 10 μL of a partly inactivated m-calpain solution. A negative relationship was observed between added inactivated m-calpain and measured calpastatin activity which suggests that the inactive m-calpain molecules mask some of the binding sites on calpastatin and thereby prevent some of the active m-calpain molecules from binding to calpastatin. This would underestimate the measured calpastatin activity. In conclusion, the calpains as well as calpastatin are stable during frozen storage of meat, and the observed decreased in calpastatin activity is due to instability of the m-calpain stock solution used in the calpastatin assay.